Gel compositions

ABSTRACT

The present invention relates to a gel composition, comprising first and second gel-forming moieties which bind reversibly to one another to form a gel. The binding of the moieties is sensitive to the level of an analyte, and either or both of the gel-forming moieties are attached to cross-linked particulate entities such that the interstices between the entities allow gel-sol and sol-gel transformation, and yet are not so small that the analyte cannot diffuse therethrough. The invention also provides drug delivery systems and sensors for detecting an analyte utilizing such a gel.

BACKGROUND OF THE INVENTION

The present invention relates to gel compositions which comprisegel-forming moieties which bind to one another to form a gel. Thebinding of the gel-forming moieties is dependent on the level of aspecific analyte and may be reversible. Such gels find use in monitoringthe level of the analyte in a sample and delivering drugs in response toabnormal levels of the analyte.

In the prior art, WO 93/13803 discloses a gel which, in one embodiment,comprises dextran and concanavalin A (ConA). Terminal glucose residuesof dextran bind to ConA, resulting in the formation of a higherviscosity gel. Reversal of this binding occurs when free glucosecompetes with the dextran glucose residues for binding to ConA. Thus,the gel is sensitive to the amount of free glucose with which it isbrought into contact. As such, the gel can be used as a drug deliverysystem using an anti-hyperglycaemic drug such as insulin. In normallevels of glucose, dextran binds ConA and the gel retains the insulintherein. However, when the level of glucose rises, the degree of bindingfalls, releasing the insulin. In a physiological situation, the releaseof insulin will result in the level of glucose falling and the degree ofbinding will increase, thereby preventing release of further insulin.Thus, the drug delivery system forms a “closed loop” system which hasthe same effect as a normally-functioning pancreas, with insulin beingreleased when required (when glucose causes the gel to undergo gel-soltransition) and retained when not required (when the lack of glucosecauses the gel to undergo sol-gel transition).

Similar gels are disclosed in Obaidat & Park, Biomaterials 18 (11 1997):801-806; Obaidat & Park, Pharmaceutical Research 12 (9 SUPPL 1995);Obaidat & Park, Pharmaceutical Research 13 (7 1996): 989-995; andValuev, et al, Vysokomolekulyarnye Soedineniya Seriya A & Seriya B(1997): 751-754.

A gel of this type is disclosed for use as a glucose sensor inDE-A-4203466. The gel is located in a semi-permeable tube such that thechange in viscosity of the gel resulting from the reversible gel-solchange causes the gel surface to respond to an oscillation signal. Thedegree of response depends on the viscosity of the gel and hence theconcentration of glucose. Later work describes the response of such agel to glucose as measured by surface plasmon resonance (SPR) to measurethe kinetics of the response rather than the viscosity itself(Ballerstadt & Schultz, Sensors and Actuators B Chemical, (1998), 46:557-567). Rather than producing a gel, a lectin was immobilised on asurface and the displacement of fluorescent labelled lectin was measuredwith laser optics. Thus, this later work did not use the change inviscosity as a measure of glucose levels. Similarly, DE-A-4034565describes the measurement of radioactive lectin from cross-linkeddextran beads.

Finally, WO99/48419 describes the use of a viscosity-sensitive chip tomeasure the change of viscosity of a reversible gel (specifically aFicoll-ConA gel) to determine the level of glucose in a sample to beanalysed.

A problem of gels of this type is that they are water-miscible and henceare prone to dispersal. This problem becomes particularly acute if theyare used in vivo to detect a particular analyte, because the componentsof the gel could cause an unwanted immune reaction: indeed, ConA ismitogenic. For this reason, the gels may be enclosed by a semi-permeablemembrane which will allow passage of the analyte into contact with thegel. However, in order for the gel to react to changed levels of analytewithin a reasonable time, the analyte must be able to pass quicklythrough the semi-permeable membrane. In addition, in those situationswhere a drug is to be released, the membrane must allow quick release.To ensure such quick passage, the membrane needs to have relativelylarge openings and/or be relatively thin, with the result that thecomponents of the gel can leach through the semi-permeable membrane. Forexample, a gel of the type described in WO 93/13803 is water-miscibleand must be confined in a small pore membrane to prevent rapid dispersalof the gel-forming components, especially in the sol form induced byraised levels of glucose. However, pore sizes (e.g. 0.1 μm) which arenot rate-limiting for release of the 36 kD insulin hexamer still allowthe 10 kD ConA tetramer and, to a lesser extent, dextran to escape.

It is therefore desirable to produce a gel of the type described abovewhich is less prone to dispersal.

According to the present invention, there is provided a gel compositioncomprising first and second gel-forming moieties which bind reversiblyto one another to form a gel, wherein said binding is sensitive to thelevel of an analyte, and either or both of the gel-forming moieties areattached to cross-linked particulate entities such that the intersticesbetween the entities allow gel-sol and sol-gel transformation, and yetare not so small that the analyte cannot diffuse therethrough.

DESCRIPTION OF THE DRAWINGS

The following detailed description of the invention will be betterunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there are shown in the drawings,certain embodiment(s) which are presently preferred. It should beunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities shown.

The invention will be described with reference to the accompanyingdrawings, in which:

FIG. 1 illustrates the polymerisation of methacrylate derivatives ofdextran and con A;

FIG. 2 is a diagrammatic representation of a dextran-con A copolymerresulting from the polymerisation of methacrylate derivatives of dextranand con A;

FIG. 3 illustrates how a matrix can be formed when at least one of thecomponents of the matrix is at least trivalent;

FIG. 4 is a diagrammatic representation of a gel in accordance with thepresent invention;

FIG. 5 is a diagrammatic representation of another gel in accordancewith the present invention;

FIG. 6 illustrates the carbodiimide addition of amine containing entity,such as con A, to a carboxyl bearing carrier, such as carbomer;

FIG. 7 illustrates structures and insulin diffusion pathways before andafter grafting con A to carbomer particles surfaces;

FIG. 8 is a graph illustrating that addition of glucose to an aqueousgel formulated with dextran and con A leads to a loss in viscosity;

FIG. 9 is a graph showing that the addition of glucose tocarbomer-containing gels formulated with dextran and con A leads to aloss in viscosity in the same range of glucose concentrations as anaqueous formulation;

FIG. 10 is a graph showing that the addition of glucose to acarbomer-containing gel formulated with dextran and con A leads to aloss in viscosity at both 20° C. and 37° C.;

FIG. 11 is a graph illustrating the viscosity reduction on addition ofglucose for carbomer-containing gels formulated with dextran and con A,and an aqueous gel formulated with dextran and con A, shown as apercentage of the original viscosity;

FIGS. 12 a and 12 b are graphs which illustrate the release of proteinfrom a gel in accordance with the present invention;

FIGS. 13 a and 13 b are graphs illustrating release of ConA frompolymerable methacrylated derivatives of ConA having been subjected tovarying degrees of polymerisation;

FIG. 14 is a graph illustrating insulin release by a copolymerised gelaccording to the present invention in the presence or absence ofglucose; and

FIGS. 15 a-c are graphs illustrating the loss of tangent values for acomposition of the present invention in the absence of chymotrypsin at20° C., 32° C. and 37° C. respectively, and FIGS. 15 d-f are graphsillustrating the loss of tangent values for the composition in thepresence of chymotrypsin at 20° C., 32° C. and 37° C. respectively,

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a reversible gel in which the interactiveligand pair (the first and second moieties) are retained within the gel(i.e. their leach to the surroundings is prevented). In addition, thephase separation of the components during the liquid stages of thegel-sol cycles is prevented, thus ensuring the juxtaposition of thefirst and second moieties and increasing the life of the composition.Preventing phase separation is also important because it fosters thecontinuing probability of interaction between the relevant ligands.

Preferred particulate entities are polymers which are locallycross-linked such that they form particles, sometimes known as“microgels”, “minigels” or “fuzzballs”, in which each particle is adiscrete hydrogel structure which is substantially or completelysurrounded by an aqueous interstitial region. It is preferred if thedegree of cross-linking is greater than that in Carbopol 941, which has1 linker for every 3300 monomers (Carnali & Naser, 1992, Colloid PolymerSci. 270: 183-193).

In certain embodiments of the invention, the particulate entities have amean diameter of 150 or 100 μm or less, preferably in the range of from10 to 80 or 20 to 70 μm, as this may prevent the composition fromcrossing a membrane with pore sizes of 0.1 mm and above, and yet allowseasy passage of the analyte and, when present, a drug which may have topass through the composition.

In a preferred embodiment, the cross-linked polymer is an acrylic acidpolymer or copolymer and is preferably a cross-linked carbomer (alsoknown as Carbopol (BFGoodrich (USA), polycarbophil, carboxyvinyl orcarboxypolymethylene polymer).

Acrylic acid is a small molecule with a reactive double bond capable ofpolymerising into linear arrangements with pendant ionisable carboxylgroups remaining from each monomer. This produces hydrophilic productscapable of behaving as polyelectrolytes. The linear derivatives of thistype (such as Carbopol 907) are reported as having an average molecularweight of 500,000 and are water soluble. Various degrees of crosslinking can also occur. Both the degree and type of cross linking hasgiven rise to a large range of polymers, many of which incorporate othermonomers besides the acid. For example, some Carbopols and Pemulens(BFGoodrich) consist of acrylic acid copolymerised with long chain alkylresidues and are cross linked with allyl pentaerythritol. Polycarbophils(marketed as Noveon) are homopolymers of acrylic acid loosely crosslinked with divinyl glycol.

Cross-linked carbomer homopolymers are characterised by being crosslinked to various extents with polyalkyl polyethers. Some arepharmaceutically more-acceptable either because of the solvent systemused in polymerisation or because they contain dispersal facilitators,but the intended utility is a function of cross link characteristics asdescribed. Most types are produced as dry powders which compriseparticles which maintain their integrity when dispersed in water. Eachparticle is an agglomerate of inseparable smaller components but themolecular weight of the primary particles is, in effect, in billions,with each linear chain being connected by the cross linker arrangements,to many others, forming one large molecule. The average diameter of thedry aggregate is generally 2-7 μm, which increases tenfold as thestructures swell on hydration and neutralisation. The latter processproduces a gelatinous product which appears to be homogeneous but infact comprises the individual swollen particles separated by aqueousinterstitial regions.

Dissolved material percolates both through and between the gelatinousparticles of the gel. Permeability of a solute through carbomer geldispersions is a function of the cross-linking density because the moretightly meshed particle interiors exclude diffusing solutes on a sizebasis much like a conventional beaded gel permeation system likeSephadex, except that the beads are smaller and less rigid. Manysubstances travel through carbomer gels much faster, therefore, thanthrough other homogeneous gels, such as hypromellose, even whenmacroviscosity is matched. Carbomers have therefore been used widely tocontrol the delivery of many solutes through a hydrated layer and areincluded in many tablet formulations for this purpose often ascomparisons with linear polymers which release by erosion. In addition,carbomers have been found to adhere strongly to mucus and have beeninvestigated for binding drug delivery structures to the gut, the eyeand the nasal mucosa in order to prolong dosage.

Preferred carbomers are Carbopol 974P, which has a tightly cross-linkedstructure (with allyl pentaerythritol) causing each hydrated particle tobe relatively rigid and to remain discrete from its neighbours, andCarbopol 934P, which is less cross linked (with allyl sucrose). Becausethese carbomers are cross-linked extensively but regionally, they areparticulate; even when they are hydrated they form flexible, small(20-70 μm in H₂O) beads. These beads will not allow the diffusion oflarge molecules through them but only around them.

Cross-linked carbomer particles have many qualities that make themattractive as carriers for proteins and other biologically activemoieties.

-   -   the particles have pendant carboxyl groups, many of which are        externally oriented. These enable the covalent attachment of        active entities including proteins, peptides and other agents        bearing active moieties such as amine groups by generic and        specific means including carbodiimide chemistry.    -   the particles are polyelectrolytes and are highly ionised at        neutral pH, enabling electrostatic attachment of suitable        cationic ligands.    -   some proteins have non-specific attraction for polyacrylic acid        and hence may not have to be covalently attached. For example,        at low pH (<4), there is a very strong physical attachment        between concanavalin A and carbomers which needs no covalent        bonding (mixtures of solutions form a precipitate) and which is        independent of the glucose receptor. Because of the low pH        required, it is preferred that one or both of the gel-forming        moieties is/are attached covalently to the particles. In a        preferred embodiment of the invention, cross-linked carbomers        are used as carriers in concanavalin A/dextran gels as an        additive. This produces a clear gel which responds very well and        yet effectively prevents the leach of protein as outlined above.    -   the covalent and electrostatic attachment of ligands can be in        very large numbers, again because of the high incidence of        carboxyls.    -   the polyanionic nature of carbomer means that it can be        dehydrated in low pH media. This allows it to be centrifuged and        thus separated from extraneous material left from coupling        procedures. It can be repeatedly washed and spun in this form        and finally rehydrated by neutralising in suitable buffer or        other aqueous media.    -   carbomers can carry certain types of solutes inside the particle        in addition to the same or other entities being present on the        external surface. Thus, for example, a therapeutic agent can be        held within the particle, whether bonded there or not, while a        targeting and/or stealth-type entity could be grafted onto the        exterior surface.    -   carbomers are very large by comparison with other carriers and        although unsuitable for some therapeutic purposes for this        reason (for example it would not extravasate), becomes more        potentially useful for others. Thus, following delivery, the        conjugate may remain longer at the target site as excretion,        metabolism and degradation proceed more slowly than with smaller        entities. Such a carrier could, for example, have much in common        with liposomes, which are of similar size and have long been        used as experimental drug delivery agents. Carbomers offer a        greater capacity for covalent attachment than is the case for        liposomes, however. As with liposomes, carbomers could be        modified covalently by the addition of polyethylene glycol        (among other hydrophilic neutral polymers) to provide a surface        with steric and other properties which prevent uptake by        macrophages in the large vessels of the liver and spleen.    -   being particulate, cross-linked carbomers can function in the        manner of an exclusion gel and therefore many solutes percolate        around rather than through the particles (or beadlets) of        hydrogel. Diffusion through carbomer gels is therefore governed        by the microviscosity of the aqueous medium in the interstitial        regions and is much faster for many solutes than for similar        consistency but homogeneously cross-linked hydrogels, where the        solute migration is a function of the macroviscosity. Where        beadlets have then been surface-modified as carriers for        covalently bound ligands, the ligands can affect the        microviscosity of the interstitial regions. This region can        therefore interact with solutes chemically or physically. Thus,        a gel bed could act as an affinity substrate for solutes in        designs for chromatographic separation, substrate sensors and        intelligent delivery devices.

There is a number of variations in which the first and second moietiescan be attached to the particles. For example, the particles can haveeither the first or the second moieties attached (covalently orotherwise) directly thereto, or can have both the first and secondmoieties directly attached (covalently or otherwise) thereto.Alternatively or additionally, instead of being directly attached, thefirst and/or second moieties can be indirectly attached via a polymer.Suitable polymers are known to the skilled person and include acrylicbackbone polymers, dextrans, celluloses and other sugar polymers. Thefirst and second moieties can be attached to each other (e.g. in themanner described in WO95/01186) with one or other being directly orindirectly attached to the particles.

The first and second moieties can be any moieties which can bindreversibly together to form a gel. It is preferred if the first moietyis a macromolecule which, when bound together, forms a gel and thesecond moiety is a molecule which binds to at least a part of themacromolecule to provide such binding. However, both the first andsecond moieties could contribute equally to gel formation. Preferably,the sensitivity of the gel to the level of said analyte arises becausethe second gel-forming moiety also binds to the analyte. Thus, theanalyte competes with the first gel-forming moiety and, when theconcentration of the analyte is sufficiently high, will prevent bindingof the first and second gel-forming moieties, resulting in a decrease inthe viscosity of the gel. As is described in more detail below, thisdecrease in viscosity can be used to release a drug or to provide anindication of the level of the analyte.

Bonding between the first and second moieties is caused by non-covalentforces such as hydrophobic, ionic, hydrogen bonding forces and the like.These interactions have been well studied in the art and their effectson molecular affinity and recognition are described, for example inKorolkovas et al, “Essentials of Medicinal Chemistry”, pp 44-81 Wiley,1976. Such reversible interactions are exemplified by the interactionbetween an enzyme and its substrate or a competitive inhibitor thereof;and antibody with its antigen, or a drug receptor site and its drug.

The second moiety may be any of a number of well-known entities whichexhibit molecular recognition and reversible binding of micro- ormacromolecules. The second moiety may be a natural binding protein, suchas an antibody, an enzyme, a regulatory protein, a drug receptor site orthe like. It is also possible to use synthetically modified bindingmolecules, such as chemically modified proteins. Such modified proteinssometimes have increased or decreased affinities for their substrateswhen compared to their natural unmodified counterparts. The secondmoiety may be a receptor built by imprinting and similar techniques(Andersson, J Chromatogr B Biomed Sci Appl. 2000 Aug. 4:745(1):3-13;Bruggemann et al, J Chromatogr A. 2000 Aug. 11; 889(1-2):15-24; Haupt &Mosbach, Trends Biotechnol 1998 November; 16(11):468-75).

It is preferred is the second moiety is a lectin. Lectins arecarbohydrate-binding proteins of plants and animals with a wide varietyof specificities for carbohydrates, (Lis et al, Ann. Review ofBiochemistry, 42, 541 (1973); Goldstein & Hayes, Adv. in CarbohydrateChemistry and Biochemistry, Vol. 35, Tipson and Horton, eds. (AcademicPress, New York, 1978, pp. 128-341). For example, ConA, a Jack Beanlectin, has specificity for α-D mannopyranose and α-D glucopyranose;soybean lectins are specific for α- and β-D-N-acetylgalactosamine andα-D-galactose units, and wheat germ lectin is specific for β-D-N-acetylglucosamine. Another lectin that may be used in the present invention isthe pea (Pisium sativum) lectin. In a preferred embodiment, the secondmoiety is a lectin, and the first moiety is a gel-forming macromoleculewhich binds to the lectin and which may be a carbohydrate polymer,preferably containing glucose, fructose or mannose moieties, such asbranched starches, dextrans, mannans, and levans, or syntheticcarbohydrates such as ficoll-400, a synthetic polysucrose, or asynthetic polymer with pendant carbohydrate or sugar moieties.

In one embodiment, blue dextran is used (Sigma). This is available intwo molecular weights (40 K and 2 M), and comprises dextran covalentlybonded to reactive blue. Each dextran molecule has many dye moietiesbonded to it, and the molecule is blue and has free amine groups fromthe dye which are available for coupling. When coupling is done withblue dextran, the product is blue. This provides a qualitative andquantitative assessment of the success of coupling.

The first and second moieties may be provided in the form of acopolymer. This may be made by polymerising prepared derivatives of thefirst and second moieties. At its simplest, this can make a linearpolymer bearing both moieties. Any type of polymer backbone produced byany relevant polymerisation technique is suitable for use in thisembodiment of the present invention.

In a second aspect, the invention provides a gel composition comprisingfirst and second gel-forming moieties which bind reversibly to oneanother to form a gel, wherein said binding is sensitive to the level ofan analyte, and the gel-forming moieties are copolymerised.

In one embodiment, the methacrylate derivatives of concanavalin A anddextran (synthesised first from the raw lectin and polysaccharide, usinga reaction with methacrylic anhydride) are polymerised to make anacrylic backbone polymer, carrying the concanavalin A and dextran aspendants (see FIG. 1).

Dextran is capable of methacrylate derivatisation (i.e. in thepre-polymerisation stage with methacrylic anhydride) at many pointsalong its length, the number depending on conditions, since eachhydroxyl group of every glucose unit in the dextran chain, ispotentially susceptible to methacrylation. Accordingly, dextran moietiescan permanently cross link the linear copolymer, producing a range ofthree-dimensional networks simply because it can start forming polymerchains at any point at which it has a methacrylate modification. Unlessthe degree of cross linking is very high, the ensuing products arelikely to be flexible and gelatinous because of the length and mobilityof dextran. Concanavalin A can also be multi-methacrylated, but becausethis molecule is globular, the product may be an aggregate and not a gelin which flexible networking extends throughout (see FIG. 2).

The fundamental character of products made by a polymerisation processsuch as the one described above is hydrophilic, but, in cases where thepolymerisation product becomes too large and complicated to remainsoluble, the product merely swells in water and does not form a solution(soft contact lenses are made from a non-derivatised version of such anacrylic). The permanent links dictate the major characteristics of theproduct in terms of its viscoelastic qualities, and so those productswhich have less derivatisation of the dextran and concanavalin A (forexample) will be viscous liquids, while those which have extensivemodification and thus allow complicated cross linking, will be solidhydrogels.

However, the interactive ligands are able to connect across the polymerchains non-covalently producing temporary bonding additional to anypermanent bonds made during polymerisation (see FIG. 2). It is thesewhich are crucial in terms of the reversible binding of the gel because,when in contact with the analyte, e.g. free glucose, the temporary bondswill be dismantled. When this happens, there will be a change in theproperties of the product and it will become more permeable, as thenotional pores throughout the lattice open up and leave only thepermanent cross links. If the derivatisation and consequent permanentcross linking of the gel has been appropriately low, a viscous liquidcan result when all of the permanent and temporary linking is in place.When the analyte is added, this liquid will lose viscosity and, becausethe reaction is reversible, this gel-sol change can be dependent on theconcentration of analyte that has diffused into the gel.

Where the first and second gel-forming entities are not copolymerised,each component is multivalent in order that a three dimensional networkor matrix results (and at least one component must have a valencygreater than two, since two divalent interactants produce a lineararrangement). This is illustrated in FIG. 3.

In a gel comprising lectin and dextran which are not attached to oneanother or to other particles, the lectin is in its naturallytetravalent form which can dissociate into stable dimers at some pHvalues. These dimers are obviously smaller and are at a greater risk ofloss from the gel. The combination of the tetravalent concanavalin A andthe multivalent (branched) dextran produces a gel, which consists of athree dimensional network stabilised with only temporary bonds. However,the components can gradually leach away when in the sol state: phaseseparation may not be obvious but may contribute to progressive loss ofaction after several cycles.

However, when the interactive components are copolymerised or attachedto particles, each component could be monovalent, and this would stillform a gel, as illustrated in FIG. 4. Accordingly, the gel of thepresent invention does not require first and second gel-forming moietieswhich are multivalent. In a preferred embodiment, lectin dimers (ortetramers stabilised by binding onto the framework) can be used. Dextranmay be substituted with a variety of other glucose bearing entities,including simple pendant glucose. However, single pendant glucose mayreduce the flexibility of the resulting gel and in fact, some permanentcross linking with dextran might be remain useful to give flexibilityand prevent leaching away in the sol phase. This is shown in FIG. 5.

As mentioned, the second moiety may be an antibody. Antibodies can beprepared and purified from animals in standard fashion (Eisen, H. N.“Immunology”, Harper & Row, 1974), and have the advantage of beinginduceable in an animal by challenge with an appropriate antigenicagent. Since this agent can be chosen from any chemical family e.g.,amino acids, carbohydrates, their respective polymeric derivatives, orthe like, the resulting antibodies may have a wide range of bindingspecificity and affinity.

Polyclonal antibodies can be raised by stimulating their production in asuitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goator monkey). Monoclonal antibodies can be produced from hybridomas. Thesecan be formed by fusing myeloma cells and spleen cells which produce thedesired antibody in order to form an immortal cell line. This is thewell known Kohler & Milstein technique (Nature 256 52-55 (1975)).Techniques for producing monoclonal and polyclonal antibodies which bindto a particular protein are now well developed in the art. They arediscussed in standard immunology textbooks, for example in Roitt et al,Immunology second edition (1989), Churchill Livingstone, London. Inaddition to whole antibodies, the present invention may use derivativesthereof, including antibody fragments and synthetic constructs. Examplesof antibody fragments and synthetic constructs are given by Dougall etal in Tibtech 12 372-379 (September 1994). Antibody fragments include,for example, Fab, F(ab′)₂ and F_(v) fragments (see Roitt et al [supra]).F_(v) fragments can be modified to produce a synthetic construct knownas a single chain F_(v) (scF_(v)) molecule. This includes a peptidelinker covalently joining V_(h) and V₁ regions which contribute to thestability of the molecule.

Other synthetic constructs include CDR peptides. These are syntheticpeptides comprising antigen binding determinants. Peptide mimetics mayalso be used. These molecules are usually conformationally restrictedorganic rings which mimic the structure of a CDR loop and which includeantigen-interactive side chains. Synthetic constructs include chimaericmolecules. Thus, for example, humanised (or primatised) antibodies orderivatives thereof could be used. An example of a humanised antibody isan antibody having human framework regions, but rodent hypervariableregions.

An example of a suitable antigen and antibody is angiotensin (Peeters,et al, J Immunol Methods 120 (1 1989): 133-43) and an anti-angiotensinantibody (available from Sigma, for example). A gel formed by thesewould be responsive to free angiotensin, which is involved inhypertensive crises. Thus, the gel could be used to monitor levels offree angiotensin and/or govern the release of angiotensin conversionenzyme (ACE) inhibitors, such as enalapril, which would treat thehypertensive crises, avoiding the need for continuous medication.

Some hormone-dependent tumours are treated with hormone antagonists liketamoxifen and cyproterone. The automatically-regulated delivery of suchdrugs in response to endogenous hormone peaks might offer an alternativeto either their blanket use, which is strongly linked to toxic effects,or to the use of agents such as goserelin (which interfere with naturalfeedback in hormone biosynthesis and can paradoxically increase symptomsby doing so). Thus, a gel formed from testosterone and anti-testosteroneantibody (Miyake, et al, Chem Pharm Bull 38 (4 1990): 951-5) could beused to deliver cyproterone for prostate cancer.

Another example is morphine and anti-morphine antibody (Kussie, et al, JImmunol 146 (12 1991): 4248-57) which could used to deliver a morphineantagonist in response to influxes of exogenous morphine-likesubstances, such as heroin, in default of addiction therapy.

The gel of the first and second aspects of the invention may be providedin combination with a semi-permeable or permeable membrane. Suitablemembranes may be of the dialysis type, e.g. with molecular weightcutoffs of 10K-500K or may be microfiltration type membranes, e.g. withpore sizes of 0.025 to 0.1 μm which may be of cellulose orpolycarbonate. The former membranes are preferred when the gel of thepresent invention is used as a sensor and the latter are preferred whenthe gel is used in a drug delivery system.

The gel-sol transition of the gel composition of the first and secondaspects of the invention in response to raised levels of the analyte canbe used to release a drug, preferably which acts to lower levels of theanalyte. Thus, according to a third aspect of the invention, there isprovided a drug delivery system comprising a gel composition inaccordance with the first or second aspects of the invention and a drug,the drug being contained either (a) within the gel composition or (b) ina reservoir with the gel composition forming a barrier between thereservoir and the area to which the drug is to be released.

In the third aspect, “drug” is intended to mean any active agent, thedelivery of which has a desired therapeutic or prophylactic effect.

As mentioned previously, the binding of the first and second gel-formingmoieties, and hence the viscosity of the gel composition, is sensitiveto the level of an analyte. This change in viscosity can be used tocontrol the permeability of a solute within it. Thus, gel compositionsof the present invention can form a closure to a reservoir or containercontaining a drug. Release of the drug from the container is governed bythe viscosity of the gel, i.e. the level of the analyte. Alternatively,the drug can be contained within the gel itself. It is preferred if thegel composition is sensitive to glucose (e.g. a ConA/dextran-based gel)and the drug to be released is an anti-hyperglycaemic drug such asinsulin. It is also possible for a glucose-sensitive gel to be used tocontrol the release of any drug, the release of the drug beingcontrolled by the administration of glucose to the patient.

When the drug delivery system of the present invention is used todeliver insulin, it is preferred if it is used intraperitoneally becausethis allows glucose to reach the system quickly and for insulin to bereleased quickly, than say if the system were used subcutaneously. Inaddition, peritoneal fluid has a glucose level which mirrors bloodglucose levels. That is not to say that the system cannot be usedsubcutaneously or even externally—the location should be selected so asto suit the condition to be treated and the drug to be released.

According to another aspect of the invention, there is provided a sensorfor detecting the level of an analyte, the sensor comprising a gelcomposition in accordance with the first or second aspect of theinvention and means for detecting the viscosity of the gel.

The change in viscosity of the gel compositions of the present inventionin response to altered levels of analyte means that they can be used tomonitor analyte levels.

The viscosity of the gel may be detected in the manner described inDE-A-4203466, Ballerstadt & Schultz, Sensors and Actuators B Chemical,(1998), 46: 557-567, DE-A-4034565 or WO99/48419.

In order to be able to monitor changes in analyte levels in close toreal time, the analyte must be able to diffuse easily through the gelcomposition and, when present, the membrane. As mentioned previously gelcompositions of the present invention allow this without escape of thegel components. In order to increase response times further, it isdesirable to detect the viscosity of a very thin (e.g. 4 mm or less,preferably 0.1-2 mm, although a monolayer is also contemplated withinthe invention) layer of gel. In such instances, it is particularlyimportant to prevent loss of gel.

According to a further aspect of the invention, there is provided amethod for the production of a gel composition of the first aspect ofthe invention, the method comprising:

-   -   causing first and/or second gel-forming moieties to become        attached to cross-linked particulate entities, the first and        second gel-forming moieties being capable of binding reversibly        to one another to form a gel, wherein said binding is sensitive        to the level of an analyte, and the interstices between the        entities allow gel-sol and sol-gel transformation, and yet are        not so small that the analyte cannot diffuse therethrough.

In a still further aspect, the invention provides a pharmaceuticalcomposition for transdermal administration of an active agent, thecomposition comprising a carrier which either (a) contains the activeagent or (b) forms a barrier between the active agent and the area towhich the active agent is to be released, wherein at least a part of thecarrier is digestible by one or more skin enzymes such that the activeagent is released from the composition by the action of said enzymes.

The invention also provides (a) the use of such a composition inmedicine, (b) the use of such a composition in the manufacture of amedicament for transdermal administration of an active agent, and (c) amethod for administration of an active agent, comprising applying to theskin of a patient in need of such administration such a composition.

In this aspect of the invention, a therapeutic agent can be releasedvariably but in a controlled manner: the intensity of medication isappropriate for the seriousness of the symptoms appertaining at thetime, the trigger for increased release being a function of the skincondition. The composition is particularly useful for releasing a drugused in the treatment of skin diseases, particularly inflammatory skindiseases, which are known to be of an inconstant nature and havesequelae of under- and over-mediation using acknowledged drugs. Forexample, in the course of psoriasis, psoriatic stratum corneum may bemore permeable when quiescent than in its thickened plaque state (Tang,et al. (1999). Clin Pharmacokinet 37(4): 273-87). Therefore, relying onhealing to slow uptake in this case is not a useful mechanism forself-adjustment of dose in psoriasis. Thus, a control system based onoutput from the formulation comprising the drug is in general consideredsuperior to one that relies on a physiological uptake mechanism.

The active agent is not covalently attached to the carrier, and thecarrier merely acts to contain (i.e. the active agent is dispersedwithin the carrier) or restrain (i.e. the carrier forms a barrier to therelease of the active agent) the active agent. This arrangement has theadvantage that it is widely applicable to a number of potential drugsbecause it does not require any covalent modification of the activeagent, however minor.

The carrier may comprise cross-linked particulate entities as definedabove with a linear or possibly branched polymer bonded to them so thateach entity has a fringe of polymer. The interstitial regions betweenthe particulate entities are occupied by the polymer, providing arheologically-active medium for drug diffusion. As discussed above,these regions have the ability to transport drugs through themrelatively unhindered compared to gels made with homogeneously meshedlinear polymers. The polymer affects the viscosity of these interstitialregions, reducing the diffusion coefficient of free drug compared withinterstitial regions without polymer. The action of skin enzymes digestthe polymer and thus raise the diffusion coefficient of the drug held inthe formulation. Preferably, the polymer has vulnerable points at manyplaces in its structure so that the long chain is broken into smallfragments if the enzymes are present. Carbomer itself is quite resistantto enzyme activity and in its native state is actually protectivetowards proteins vulnerable to proteolysis (Hutton, et al. (1990). ClinSci (Colch) 78(3): 265-71; Luessen, et al. (1995). Pharm Res 12(9):1293-8; Walker, et al. (1999). Pharm Res 16(7): 1074-80). Its stabilitycan maintain a minimum viscosity appropriate to a non-dripdermatological preparation, even when enzymes actively affect thepolymer, but its protective effect lessened due to the modification ofthe surface.

Clearly, the selection of the polymer depends on the ability of skinenzymes to degrade it to release the active agent. Polymers or moleculeswhich are degraded by one or more of the following skin enzymes can beselected by those skilled in the art: elastase (Chandler, et al. (1996).Biochem Biophys Res Commun 228(2): 421-9) which is a lysosomalproteinase responsible for the breakdown of elastin fibres in the ageingprocess, but also found in psoriasis; SCCE or stratum corneumchymotryptic enzyme (Ekholm & Egelrud (1999). Arch Dermatol Res 291(4):195-200), a serine protease known to participate in surface cellshedding and to be implicated in psoriasis; and LTA4 hydrolase which canproduce the abnormal leukotriene LTB4 from LTA4, a process also observedto occur in psoriasis. These three enzymes have esterase and/or amidaseactivities which are not necessarily specific for their originalphysiological substrates. It has been shown (Higuchi, et al. (1988).Inflammation 12(4): 311-34) that crusts formed in experimental lesionsare rich sources of proteases and they have also been found in sweat(Horie, et al. (1986). Am J Physiol: R691-8). Furthermore, stratumcorneum thiol protease (SCTP) is a cysteine protease recently found inthe upper regions of stratum corneum. It is gelatinolytic as measured byzymography and functions best at the slightly acidic pH of the skin(Watkinson, (1999). Arch Dermatol Res 291(5): 260-8). Collagenasesbelong to the metalloprotease (MMP) enzymes which control collagenturnover in connective tissue, organ tissue, bones and cartilage amongother locations. They are important in normal and abnormal skinincluding the healing of wounds (Simeon, et al. (1999). J InvestDermatol 112(6): 957-64). A subset, type IV, the gelatinases MMP-2 andMMP-9 plus their specific inhibitors, are found in the dermis andepidermis, often associated with the keratinocytes and the Langerhanscells for which they modulate cell migration within the matrix duringskin function (Kobayashi, Y. (1997). Immunology 90(4): 496-501; Makela,et al. (1999). Exp Cell Res 251(1): 67-78). The proteolytic activity iswell understood (Seltzer, et al. (1990). J Biol Chem 265(33): 20409-13;Seltzer, et al. (1989). J Biol Chem 264(33): 19583-6) but the role indisease less so. There is some confusion in the technical literatureabout the detail but there are always raised levels of MMPs, m-RNA andtheir natural inhibitors in inflammatory skin diseases (Feliciani, etal. (1997). Exp Dermatol 6(6): 321-7; Buisson, et al. (2000). J InvestDermatol 115(2): 213-8; Fleischmajer, et al. (2000). J Invest Dermatol115(5): 771-7). Faults in the construction of the psoriaticdermal-epidermal have been attributed to the overexpression of theenzyme and the enzyme has also been found in keratinised layers near thesurface. In psoriasis, gelatinase may be raised in those skin areas thatare particularly involved.

Preferably, the polymer is selected from soluble synthetic homo- andco-polymers of amino acids (i.e. synthetic polypeptide analogues),mucin, collagen and gelatin. These all contain amine groups so that theattachment of polymer to the carbomer carboxylate groups can beaccomplished using a standard carbodiimide method, but could also formSchiff base conjugates. Chitosan, a biodegradable polysaccharide mayalso be used.

Soluble synthetic homo- and co-polymers of amino acids need hydrophilicgroups, such as pendant amines, hydroxyls and carboxylates, to havesolubility. These compounds, such as e.g. polyaspartate andpolyglutamate, can be viscous at high molecular weight. Some havepreviously been used in drug delivery (Singer, et al. (2000). Ann N YAcad Sci 922(136): 136-50; Yi, et al. (2000). Pharm Res 17(3): 314-20)but are very expensive.

Mucin is a glycoprotein with a substantial viscosity and can bepurchased (Sigma) in various degrees of purification: Type I (the purestwith 12% bound sialic acid), type II and III (progressively more crudeand each with 1% sialic acid).

Collagen and gelatin have a number of favourable characteristics,including a relevance to skin collagenases as discussed above. Collagenitself is expensive, and therefore it is currently preferred to use itscheaper derivative, gelatin.

Gelatin is a soluble proteinaceous derivative of collagen. It has wellknown rheological properties such that it is free flowing and viscousabove a critical temperature (which in some grades is around 36° C.),and an elastic solid below this temperature, provided the concentrationis high enough. It is not toxic and has been used in topicalpreparations, at least in experimental and development work as well asfor buccal application, rectal use, dermal injections, as deep wounddressings and as an iv plasma expander. Some gelatin is of bovine originand carries theoretical risks of prion contamination but there arealternative sources The World Health Organisation has dictated thatpharmaceuticals containing gelatin should be made from supplies fromBSE-free countries (WHO/EMC/DIS/96.147).

Like the parent collagen, gelatin contains seven main amino acids (plusseveral others in small percentage). The molecular arrangement isunusual because of the proline twist which promotes the formation of thetriple helix structure in collagen. Four of the main amino acid residuescontain hydrophilic pendants to which covalent attachments can be made.The availability of groups makes gelatin suitable for interstitialfringes as described above. Indeed, it can be bonded to a carbomer viathe terminal amine groups in lysine and hydroxylysine and arginineresidues. Gelatin is degraded by collagenases as well as a variety ofother proteases, including some chymotrypsins and elastases. Theliquefaction of a gelatin substrate is used to indicate chymotrypsins instool tests for cystic fibrosis and for the identification of certainbacteria.

Active agents that can be used in this aspect of the invention for thetreatment of psoriasis include:

-   -   salicylic acid, tars, dithranol (anthralin)    -   steroids, vitamin D analogues, retinoids (vitamin A analogues)    -   psoralens, methotrexate, ciclosporin (cyclosporin)

Steroids such as mometasone and fluticasone are often prescribed aloneor in combination with retinoids such as tazarotene or with vitamin Danalogue calcipotriol. Steroids, vitamin D analogues and more recently,retinoids, have been marketed for some years as straightforward topicalapplications such as ointments, creams and gels. Patients have apreference to these treatments over tars and dithranol because they areat least as effective and easy to use (Poyner, et al. (2000). J Eur AcadDermatol Venereol 14(3): 153-8). The following will focus on theretinoids which may have particular appeal as discussed below, but thisaspect of the present invention is all three of the above groups.

Retinoids form a group of compounds of which vitamin A compounds form asubgroup and have the biological activity typified by the alcoholretinol. Retinol (1) itself is metabolised reversibly to retinaldehyde(retinal) (2) and thence to the non-vitamin A but therapeuticallyactive, retinoic acid (3)

(1) R = CH₂OH (6) R = CH₂NH₂ (2) R = CHO (7) R = CH═NOH (3) R = CO₂H (8)R = CH═N[CH₂]₄CHNH₂CO₂H (4) R = CH₃ (9) R = CO₂C₂H₅ (5) R = CH₂OCOCH₃

The retinoid nomenclature is often stretched to include otherbiologically related materials such as the arotinoids, of which therecently introduced agent tazarotene is a further modified analogue,used for treatment of psoriasis. Adapalene is pharmacologically similarbut structurally distant.

The retinoids are important in the transmission of visual signals, andin the growth and differentiation of cells. The stereochemistry of thesecompounds importantly involves the polyprenoid side chain that can beall-trans or exhibit various cis isomerisations. Chiral centres alsoexist in the ring at the 1 and 3 positions. The stereochemistry isimportant for some of the biological activity of retinoids. In themammalian retina, for example, the protein rhodopsin, is reversiblytransformed by light as its aldehydic chromophore retinal convertsbetween the all-trans and 11-cis forms.

In the skin, some retinoids, including retinoic acid isomers (Vahlquist,(1999). Dermatology 1(3): 3-11) and their relatives, activate particularnuclear receptor types in keratinocytes, some of which may bestereosensitive, triggering gene expression. This has at least threeeffects including the production of mRNA and protein pertinent toreceptor induction, proliferation of epidermis and changes in thekeratinisation processes (Didierjean, et al. (1999). Exp Dermatol 8(3):199-203). The mechanism of action of these and related compounds in thetreatment of psoriasis is, however, not clear (Saurat, (1999). J Am AcadDermatol: S2-6) and the evidence is somewhat paradoxical. First, thereis apparently no correlation between the observed binding to thereceptors and therapeutic activity such that acetretin activatesreceptors without binding while tazarotene shows differential binding tothree receptor subtypes. This may be a temporary confusion resultingfrom the variations within the three receptor subsets. The second puzzleis that lesions already show increased retinoic acid (isotretinoin)formation (Arechalde & Saurat (2000). Biodrugs 13: 327-333), yet orallythis compound and acitretin (an ester) work well and have been shown tocontrol cell differentiation and sequential metaplastic changes. Recentwork points to a decreased mRNA expression in lesional skin followingreceptor activation (Torma, et al. Acta Derm Venereol 80(1): 4-9),possibly partially explaining this finding. Oral isotretinoin andacitretin (a recent replacement for etretinate) have been used for morethan two decades to treat psoriasis but involve a significant list ofcontraindications and precautions. The application of retinoic acidtopically is used to treat acne and not listed in the BNF forantipsoriatic activity. However, the introduction of tazarotene (Tang,et al. (1999). Clin Pharmacokinet 37(4): 273-87) for this indicationshows that it may feasible to use other retinoids topically forpsoriasis, despite the receptor subset specificity claimed fortazarotene, which also has an excellent safety profile (Marks, R.(1998). J Am Acad Dermatol: S134-8). However, it is reported that newreceptor subset-specific retinoids are under development possibly as anantidote to the mucocutaneous toxicity of oral isotretinoin (Nagpal &Chandraratna (2000). Curr Pharm Des 6(9): 919-31). Rosacea (Vienne, etal. (1999). Dermatology 1(53): 53-6) and photodamage (Katsambas &Katoulis (1999). Adv Exp Med Biol 455(477): 477-82; Sorg, et al. (1999).Dermatology 1(13): 13-7) may also respond to topical retinoids, whileretinoids delivered topically may protect skin against the atrophiceffects of topical steroids (McMichael, et al. (1996). Br J Dermatol135(1): 60-4). The applications extend as tazarotene is also used foracne, as is adapalene.

Locally applied retinoic acid is less toxic than oral retinoids butnevertheless produces irritation and erythema. However, use of thealdehydic prodrug retinal, has been reported as particularly attractivebecause it is less aggravating and exerts its effects by conversionwithin keratinocytes, to active species, after which receptorinteraction occurs (Didierjean, et al. (1999). Exp Dermatol 8(3):199-203; Sorg, et al. (1999). Dermatology 1(13): 13-7). Much isesterified and acts as a storage reservoir. The remainder is metabolisedto small quantities of bioactive alcohol and acid forms. The latteroccurs at a rate dependent on the oxidation capability of cells, whichitself is related to the differentiation and thus possibly overt diseasestatus of the cell population (Sorg, et al. (1999). Dermatology 1(13):13-7). In terms of biological results, the all-trans retinal, which ismetabolised to the all-trans retinoic acid (tretinoin) was foundsuperior to a 9-cis analogue, which is likely to have formed thecorresponding retinoic acid isomer (Didierjean, et al. (1999). ExpDermatol 8(3): 199-203).

The retinoids are known to be teratogenic. Retinoids are ubiquitoussignalling molecules and their deficiency and inappropriate availabilityboth cause developmental faults. A variety of effects has beendocumented including craniofacial, limb and nervous system defects(Kubota, et al. (2000). Eur J Pediatr Surg 10(4): 248-51). The majorrisks may be for oral preparations but cannot be ignored for topicals,although tests with an animal model have used many times the topicalconcentration of isotretinoin without harm to the foetus.

The retinoids are in formulated products as follows:

Tigason (Roche) etretinate Discontinued Neotigason (Roche) acitretin 10mg oral Roaccutane (Roche) isotretinoin (13-cis retinoic  5 mg oralacid) Isotrex (Stiefel) isotretinoin  0.05% gel (for acne) Retin A(Janssen tretinoin (all-trans retinoic  0.01% gel (for acne) Cilag)acid) 0.025% cream (for acne) 0.025% lotion (for acne) Retinova(Janssen-  0.05% cream (for acne) Cilag) Zorac (Bioglan) Tazarotene 0.05% gel (for acne and psoriasis)  0.1% Differin (Galderma) Adapalene 0.1% gel (for acne)

The retinoids are very hydrophobic compounds. The water solubilities atroom temperature and pH 7.3 buffer (with antioxidants) are documented asfollows (Szuts & Harosi (1991). Arch Biochem Biophys 287(2): 297-304):

retinol 0.06 μM retinal 0.11 μM retinoic acid 0.21 μMi.e. they lie between 0.000002 and 0.000006% w/v.

When used in this aspect of the invention, there must be sufficientwater solubility of the drug to make it feasible to deliver an effectivedose. The solubility figures above imply for example that the 0.01% w/vtretinoin gel in the list must contain a co-solvent. Retinoidpreparations such as Isotrex and Acticin contain ethanol while otherssuch as Differin contain propylene glycol and poloxomer, all inconjunction with gelling polymers such as hydroxypropylcellulose andcarbomers. Alternatives are to formulate the gel with surfactants, suchas the polysorbate 40 in Zorac and the technical literature showscomplexation of retinoic acid with cyclodextrin for iv administrationand other uses (Botella, et al. (1996). Journal Of Pharmaceutical AndBiomedical Analysis 14: 909-915; Lin, et al. (2000). J Clin Pharm Ther25(4): 265-9). A liposomal or similar lipid aggregate component couldalso function in a gel to increase the colloidally dispersedconcentration (Li, et al. (1999). Photochemistry And Photobiology 69:500-504).

Thus, in accordance with one embodiment of this aspect of the invention,a carbomer-gelatin conjugate can be used to deliver a retinoid in a skinenzyme-dependent manner. The retinoid may be conventional tretinoin,tazarotene or retinal, which is an inactive prodrug converted in situ.All of these have low solubility but may be solubilised in cosolvents,surfactants or liposomes.

Retinal and retinoic acids (isotretinoin and tretinoin) have groupswhich allow their conversion to conjugates. This can not only increasetheir water solubility but provide an enzyme-dependent release whichcould be used per se or alternatively in tandem with a pharmaceuticalcomposition for transdermal administration of an active agent asdescribed above.

In a still further aspect of the invention, there is provided acomposition comprising a carrier which is covalently bonded to an activeagent, wherein the bond between the carrier and the active agent isdigestible by one or more skin enzymes, for use in medicine, inparticular for transdermal administration of the active agent.

The invention also provides a method for administration of an activeagent, comprising applying to the skin of a patient in need of suchadministration a composition comprising a carrier which is covalentlybonded to an active agent, wherein the bond between the carrier and theactive agent is digestible by one or more skin enzymes

In this aspect of the invention, the active agent is covalently bonded(directly or indirectly) to the carrier. Enzymes making contact andmixing with the composition must be capable of cleaving thisagent-carrier conjugate to release the medication. The conjugates can beconsidered to be prodrugs that undergo activating metabolism outsiderather than inside the skin. The carrier could be one of a variety ofpolymers currently used in dermatological formulation, and it ispreferable that the composition is single phase and aqueous. Polymerssuch as carbomer, polyvinylpyrrolidone or cellulose derivative, forexample, with linear or more involved construction dissolve in anaqueous medium to give suitable properties. The connection between thedrug and polymer may be direct or indirect. In the latter case, theconnection may be via a bridging molecule. This molecule may also bepolymeric in which case enzymes can release the drug by attacking bondsin the bridge. The molecule may be a peptide or polypeptide. Polymerbridges can be used to introduce enzyme specificity in conjugates usedsystemically. In either case, many drug molecules can be attached tovarious parts of the polymer structures, provided of course that thedrug is undamaged by the conjugation and cleavage.

Such conjugates are known but do not appear to have been used fortransdermal administration before. Schiffs base conjugates with retinalhave been studied in an attempt to elucidate how rhodopsin operates asan optically-activated neurotransmitter. Thus, retinal conjugated withdextran, polylysine, polyethylene glycol have been described as watersoluble conjugates, while Schiff base conjugates of retinal withphosphatidylethanolamine and with alkylamines produced micellisedaggregates (De Pont, et al. (1969). Exp Eye Res 8(2): 250-1; Adams, etal. (1974). Exp Eye Res 18(1): 13-7; Pitha, et al. (1980). J Natl CancerInst 65(5): 1011-5; Freedman, et al. (1986). Photochem Photobiol 43(3):291-5; Singh, et al. (1990). Biochim Biophys Acta 1036(1): 34-40;Viguera, et al. (1990). J Biol Chem 265(5): 2527-32). Such conjugatescould therefore confer useful water solubility.

Preferred features of this aspect of the invention are as for thepharmaceutical composition for transdermal administration of an activeagent described above.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

The invention will now be described further in the followingnon-limiting examples.

EXAMPLES Introduction

Formulations of concanavalin A (con A) and dextran have been shownpreviously to be glucose sensitive. Con A is a protein with a glucosereceptor in each of the subunits contributing to the structure. Theprotein is dimeric below pH 5.8 but tetrameric between pH 5.8 and 7.0,above which point it begins to form larger aggregates. The receptorsaccommodate free glucose but also terminal glucose units onpolysaccharides such as dextran. Because dextran is multivalent in itscomplement of such terminals, an admixture with the lectin producescomplex structures which can be either a precipitate or gelatinous. Thestructures are destabilised by the addition of free glucose whichcompetes for occupation of the receptor sites. Under circumstances whereglucose is added to a gelatinous formulation of the mixture, theviscosity falls sharply but is regained if glucose is dialysed out. Thismechanism forms the basis of a delivery device for hypoglycaemic agentssuch as insulin which would diffuse more slowly through the gel ifambient glucose levels were low but could be triggered into a higherdelivery rate if the viscosity was reduced by contact with glucose.

A problem with this design is the tendency of the components to leachaway from the gel. The components are water dispersible when the gelbecomes dismantled and some means is required by which the sol form isstabilised to prevent loss from the formulation but which will notprevent the viscosity change upon which the design depends. In thefollowing, the lectin was exposed to a procedure to bond it covalentlyto commercially-produced carbomer types Carbopol 974 and 934 (referredto as C974 and C934). These materials are polyacrylic acid derivativeswhich have been three-dimensionally cross-linked to produce gelatinous,but particulate, entities, each with a molecular weight of severalbillion. The bonding has been done with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC),which operates to give a temporary intermediate structure between theEDAC and a carboxyl moiety (in this case from the carbomer sites (seeFIG. 6)). The intermediate can then bond with amine groups, such as arefound in proteins including con A at terminal and lysine sites. Thisresults in the formation of an amide linkage and the loss of the EDACresidue. The con A can become effectively conjugated with the carbomersuch that the surface of the carbomer particles becomes permanentlycoated with lectin.

The carbomers C934 and C974 differ from many polymeric gels in that,although they are both cross-linked, neither is an integral structure.Each hydrated particle is in effect a discrete hydrogel and interstitialaqueous regions separate it from neighbouring particles. This means thatlarge molecules like insulin can diffuse through a carbomer gel of thistype more rapidly (FIG. 7 a) than through gels made from linear polymers(including linear carbomer types) or through regular hydrogels. In boththe latter, the obstructions are greater since physical and chemicalentanglements respectively occur throughout the structures and couldwell be rate-determining for insulin transport.

Reagents

-   Carbopol 974 (C974), Carbopol 934 (C934) BF Goodrich-   EDAC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride],    Sigma-   MES [2-(N-morpholino)ethanesulfonic acid], Sigma-   PBS (phosphate buffered saline, pH 7.4 & 5.9)-   NaOH (pellets and 1M solution), Fisher Scientific-   dextran (RMM 2 000 000), Sigma-   sodium azide, Sigma-   concanavalin A, Sigma-   distilled water-   1M HCl (hydrochloric acid), BDH

Example 1 Production of Gel and Measurement of Viscosity

A 1% w/w carbomer dispersion was made in 0.1M MES buffer, adjusted toneutral and stirred until clear. Con A was then added to this such thatthe final concentration was also approximately 1% w/w and the pH kept atneutral. The system was then conjugated using 50 mM EDAC, the reactionbeing quenched after stirring for three hours at room temperature bydiluting with PBS at pH 5.9 and centrifuging washings from thepartially-dehydrated carbomer conjugate until washings revealed nofurther con A in the supernatant. The total protein removed wascalculated by assaying bulked and filtered washings at 276 nm. Thecoupling efficiency was greater than 90%. A 1 g quantity of a 20% w/wdextran D2M (mw 2 million) solution in PBS (pH 7.4, preserved with 0.01%sodium azide) was then added to the neutralised carbomer-con A conjugateand mixed thoroughly. The final product therefore contains 200 mg eachof carbomer and dextran D2M. The pH of the gel was then adjusted to 7.4using 1M NaOH and a stiff, glucose responsive gel results. The finalweight is slightly variable, depending on the final pH adjustments andthe contents are calculated accordingly.

Both C974 and C934 were conjugated in this way and subjected toviscosity testing in the presence of varying concentrations of glucose.The viscosity measurements were obtained using a Haake Rheostress RS75cone and plate viscometer in continuous rotation mode where rate ofshear was ramped between 0 and 5 s⁻¹. The viscosity values correspondingto a shear rate value of 5 s⁻¹ were used to compare the gels at theglucose concentrations between 0 and 5% w/v. The gels were compared at20° C. and the C934 product was also measured at 37° C.

The conjugates produced from the particulate carbomers, C974 and C934,were calculated to have compositions as follows:

C934 (formula code E100) C974 (formula code E87-5) Carbomer 2.6% w/w2.7% w/w Dextran D2M 2.6% w/w 2.7% w/w Con A 2.4% w/w 2.5% w/w

The formulations were rather opaque in comparison with the extremeclarity of carbomer gels from which they are derived. The protocol inwhich the con A was added in the presence of excess EDAC is likely tohave produced a variety of products which might include strings oflectin added to the carbomer. This is because con A also has carboxylgroups which are vulnerable to the action of EDAC. However, this methodgave a result in which the con A was bonded in high proportion and itwas used in preference, at this stage, to one in which the carbomer-EDACreaction was quenched prior to adding the con A.

In terms of assessing the performance of the formulations, it is usefulto compare these products with simple aqueous combinations of dextran2DM and con A without carbomer. However, a direct analogy was notpossible, because 2.5% con A forms only precipitates with lowconcentrations of dextran in carbomer free aqueous admixtures.Accordingly, 2.5% con A was combined with 10% w/w D2M to form a lowviscosity gel. This concentration of dextran in the presence of carbomergave too viscous a product to be useful in the study and the dextrancontent in them is about 3% w/w.

FIG. 8 shows that the addition of glucose produces a progressive drop inthe viscosity of the aqueous formulation mainly over the glucoseconcentrations 0-0.5% w/w (highlighted on the graph), which is a rangerelevant for the design of products useful in the control of diabetesmellitus. The response is dependent on the stoichiometry of thedisplacement mechanism described above and is therefore a function ofthe relative concentrations of dextran and glucose. It is this responsewhich it is important to conserve.

The results in FIG. 9 show a drop in viscosity over a similar glucoserange to the aqueous formulation, demonstrating that glucose sensitivityhas been imposed on the carbomer particles. However, the baselineviscosity levels, at which the lectin-dextran linkages have beenreversed with glucose, are much higher when carbomer is present becauseof the viscosity of this polymer.

FIG. 9 also indicates that the addition of dextran and con A has raisedthe viscosity of the glucose-free product by a much greater amount incarbomer systems than in plain aqueous ones, despite the lower dextrancontent used in the carbomer-containing formulations. This means thatthe glucose response, in terms of the viscosity change, is of a greatermagnitude in the carbomer formulations than in the aqueous one, as isalso shown in FIG. 9. This also happens to be the case if there is nocovalent bonding of the lectin to the carbomer (not shown) and istherefore a function of the presence of the carbomer on the dextran-conA system. An explanation for this relates to the heterogeneity of thecarbomer dispersion discussed above. The dextran and con A arerestricted to the interstices because their size (mw 2 million and100,000 respectively) must exclude them from C974 and C934 particleinteriors. Their localised concentrations must therefore be much higherthan the total concentrations because the interstices form a fraction ofthe total volume. This explains the difference in the physicalproperties of the complex formed between dextran and con A at the verylow concentrations used in carbomer systems compared with entirelyaqueous systems, as described earlier. In the carbomer systems, themicroviscosity of the interstitial region clearly contributes much tothe glucose sensitivity of the formulation. However, the macroviscositychanges caused by glucose, as sensed in this experiment, do not appearto be simply an average for an active interstitial and an inertparticulate compartment because the glucose-induced changes are so muchlarger than in the homogeneous aqueous system. This seems to imply thatthe interstitial viscosity is not the sole reason for the increasedglucose sensitivity of this system but probably that its presenceinfluences the freedom of movement of the particles in the gel as awhole and thus has an additional effect on its macroviscosity.

A similar viscosity fall occurs at 37° C., as shown in FIG. 10 for theC934 conjugate. The viscosity values and the changes at 37° C. are bothlower than for 20° C. as might be anticipated from the similarbehaviours of both carbomers and the aqueous, carbomer-free formulationsof dextran and con A (neither shown). However, the system is clearly asensitive glucose sensor at physiological temperature.

In the systems examined at 20° C., the viscosity change when expressedas a fraction of the original value is lower for the carbomer systemsthan for the aqueous systems, as is shown in FIG. 11, despite the higherabsolute values.

The main reason for wishing to covalently bond the lectin to thecarbomer is to prevent its escape from the formulation withoutsacrificing activity. The viscosity changes induced by glucose showthat, after washing the carbomer several times by centrifuging thesupernatant from it, it has glucose sensitivity. This suggests that thelectin has been successfully anchored to the carbomer particle surfaceusing EDAC as the bonding agent. A plain mixture of carbomer and thelectin does not retain activity when washed in this way (not shown).

Example 2 Insulin Diffusion

The gels described in Example 1 were also subjected to insulin diffusiontests at 37° C. In these experiments, six small delivery cells wereused, each capable of holding 0.5 mL of insulin solution or a buffercontrol solution. They were each mounted empty into temperatureregulated receptor vessels, holding 10 mL of buffer and were connectedby individual flow through systems to a Perkin Elmer Lambda 40spectrophotometer, programmed sequentially to scan each receptorsolution between 250 and 500 nm at ten minute intervals. The cells werefilled by syringe at the required time and then sealed using a taparrangement to make them watertight. In each cell, a thin layer of thegel was sandwiched between two filter membranes such that the insulindiffused through the gel from the reservoir in order to reach thereceptor solution. Glucose could be added to individual receptorsolutions in the concentration required at any time during theexperiment. To remove the glucose, the receptor fluid was replacedbetween readings with new buffer at 37° C., the flow through circuitflushed and the buffer replaced again before the next reading was due.

For those diffusion cells containing insulin, the addition of glucose atthe points shown on FIG. 12 a has resulted in increased output withinabout 60 minutes of the addition of 0.5% glucose. FIG. 12 b shows theresults from buffer-containing control cells and represents the glucoseprovoked release of con A. This indicates that the con A loss from thegel layer has been minimised because, in aqueous dispersions,glucose-induced liberation similar to that of the insulin would haveoccurred (not shown). Although the loss of con A has therefore beenshown to be almost negligible, the results in FIG. 12 a havenevertheless been corrected for this effect. In addition, a correctionhas been made in both the insulin and the control systems, for opticaldensity as measured at 460 nm. The optical density is a measure ofprotein precipitation, including the production of fibrils. Fibrillationis a much greater problem with insulin than with con A in thisexperiment. It results from the application of shear forces such aswould be experienced by solutions circulating through a peristalticpump. The flow rate in the circulation system from the receptor to thespectrophotometer was kept as low as possible in this experiment toreduce the problem, consistent with maintaining the release process fromthe device as the rate-determining process. The effect needs to becompensated for by subtraction from the 276 nm profile, however, becauseit does not remain a constant throughout the experiment as the glucoseis removed by fluid replacement and because it is a non-constantfunction of protein concentration.

The result of covalent bonding of the lectin to the carbomers C934 andC974 and its combination with dextran has therefore maintained theactivity of the glucose-dependent mixture of dextran and con A in termsof controlling the diffusion of insulin as a function of glucosecontent. This has operated at a concentration of glucose that representsa fairly severely diabetic state. The method has also has created aformulation from which con A cannot leach through apertures smaller thanthat which retains carbomers in the gel layer. Since carbomers like C934and C974 have molecular weights in billions (and a particle size in themicrometer range), this effectively allows the use of very large porerestraining membranes in the set-up described in this example. Theadvantage of this is that the gel itself—and not the cellulose membranesbetween which the gel is sandwiched—will be rate-determining for theinsulin. This has been a problem in unconjugated mixtures which requirepore membranes with a molecular cut-off close to the size of insulin inorder to retain the lectin.

The data presented in Examples 1 and 2 indicate that it is possible toproduce unusual conjugates between particulate carbomers, such as C974or C934, and a second component, such as a protein, in which some of thechemical characteristics of the protein can be preserved. In this case,con A has been bonded to cross-linked carbomers to produce systems inwhich the interstitial regions of the formulations become glucosesensitive. The EDAC method has been used but alternatives are possible.If dextran is added to this conjugate, the system becomes viscousbecause of the temporary links formed between the lectin and the branchends on the dextran. The viscosity of this system is then a function ofthe free glucose content because of the competition for the glucosereceptors in the lectin. This mechanism works well in the simplemixtures of con A and dextran but operates in an enhanced way in thecovalently-conjugated formulation. This enhancement appears to berelated to the heterogeneous structure of particulate carbomer gels inwhich the lectin-dextran complexes are formed in that fraction of thesystem between particles, creating high localised concentrations of theglucose-sensitive components. These conjugated formulations have beenshown to produce glucose dependent reductions in macroviscosity. Theyhave also have been able to transmit insulin in a glucose-related mannerwithout the loss of free lectin. The latter remains covalently bound tothe carbomer particles and therefore restrained by membrane pore sizesthat need be small enough only to retain the carbomer particles, thusnot compromising the rate-determining process in this glucose dependenttransport system.

Example 3 Copolymerised Gels

Methods

Concanavalin A Methacrylation

Concanavalin A was refluxed at 50° C. for 2-3 hours with methacrylicanhydride in phosphate buffered saline at 7.4. 500 mg of ConA wasdissolved in 10 mL phosphate buffered saline in a 50 mL round bottomedflask and to it was added 0.05 mL distilled methacrylic anhydride. Afterrefluxing, the reaction was quenched with distilled water (20 mL) andthe whole solution dialysed against distilled water for 2 days to removeproducts of mw<12-14,000. This process cannot remove unbonded con A.

Dextran Methacrylation

A 10 g quantity of D500, dextran of mw 500,000, was weighed and driedover phosphorus pentoxide. Separately dimethylesulphoxide (DMSO) wasdried over calcium hydride and then distilled. A 100 mL quantity ofdistilled DMSO was then added to the dried dextran and the resultingmixture stirred to dissolve at 50° C. over an oil bath. To this wasadded 200 mg of dimethylaminopyridine (DMAP) and 2.77 mL of distilledmethacrylic anhydride. The mixture was then stirred at 50° C. on the oilbath for 24 hours under reflux. Precipitation was achieved by addingdropwise into IL acetone to give white flakes of dextran methacrylate.The product was then dissolved in 500 mL of distilled water and dialysedagainst distilled water to remove small mw products such as DMSO, DMAP,methacrylic anhydride.

uv Cross-Linking of the Methacrylated Deriatives

An initiator, Irgacure (photoinitiator, Ciba Speciality Chemicals), wasused to start a uv curing process. 20 μL of a 40 mg/5 mL aqueoussolution of Irgacure was added. The sample was then irradiated at 365 nmat 10 mWatts/cm² for a predetermined time, split for equal irradiationof both sides of a spaced film between glass plates.

The variables in the system are:

-   -   concentration of components    -   concentration of initiator    -   degree of dextran and con A substitutions    -   irradiation time

These variables allow gels of a wide variety to be made. In thefollowing case, the irradiation time was varied between 2 and 50minutes.

Diffusion Tests

The gels made as described above were then subjected to the samediffusion tests as referred to Example 2 and the results were asfollows:

Results

In FIG. 13 a, the leach from polymerable methacrylated derivatives ofthe protein con A is plotted before and after the provocation withglucose (experiment conducted at 37° C., using 0.2 μm pore size filters,and glucose trigger concentration of 1% w/v). The increase in radiationtime of a gel subjected to uv and an appropriate initiator, leads to areduction in the peak of protein produced as the gel softens on contactwith glucose. Short irradiation times (e.g. 5 min—triangle symbles) donot achieve this, whereas the 20 minute cure time (diamond symbols)produces only a slight leach. The polymerisation has therefore achievedthe aim of locking in the protein within these longer-irradiated butstill non-rigid gels. However, the comparison with the gel-free control(filled square symbols) shows that some protein release occursthroughout, albeit slight, even with 20 minute irradiation.

FIG. 13 b shows that much longer irradiation times solve this problemand 50 minutes seems to give a product that hardly loses protein at all,either before, during or after glucose addition. This is also non-rigidand thus suitable for the purpose.

FIG. 14 indicates that under these conditions of minimised componentleach, glucose can be shown to provoke a release of insulin from areservoir held behind a layer of gel in an exactly analogous set up. Thegel is therefore capable of response, such that insulin is released, butit will not leach out the component protein con A. The gel seems toreform after glucose removal, so that the insulin flux is restored topre-glucose levels. This means it is truly reversible, implying thatneither gel component is lost.

Conclusion

Polymerisation is an effective method for producing gels that are asresponsive as the plain mixtures but resist the tendency to losecomponents.

Example 4 Gelatin:Carbomer Conjugation with EDAC

Conjugation between gelatin and carbomer has been done with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC),which gives a temporary intermediate structure between the EDAC and thecarbomer carboxyl moiety. The intermediate bonds with amine groups, suchas are found in gelatin at terminal and hydroxylysine sites, amongothers. The gelatin is effectively conjugated with the carbomer suchthat the surface of the carbomer particles becomes permanently coatedwith gelatin while the EDAC residue leaves the permanent bond site. Themethod here fosters some gelatin-gelatin bonding in addition tocarbomer-gelatin bonding since carboxyls and amines both occur ingelatin. This is of no disadvantage since it means that all gelatinadded, even to quite high concentrations is likely to be bonded, evendistantly, to the carbomer carrier, which therefore may have not simplymonolayers but multilayers of gelatin bonded to each micro- (or mini-)gel hydrated carbomer particle.

However, the method can be modified to produce the intermediate (betweenthe carbomer and EDAC), wash to remove excess EDAC and then bondgelatin, in which case, the gelatin content will be minimised (monolayeronly).

Each hydrated carbomer 974 or 934 particle is a discrete hydrogel andseparated from others by interstitial aqueous regions. This means thatthe gelatin bonded to the surface can raise the viscosity of theinterstices until hydrolysed by enzyme or chemical action.

Reagents

-   Carbopol 974 (C974), Carbopol 934 (C934) BF Goodrich-   EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride],    Sigma-   MES [2-(N-morpholino) ethanesulfonic acid], Sigma-   PBS (phosphate buffered saline, pH 7.4 & 5.9)-   NaOH (pellets and 1M solution), Fisher Scientific-   sodium azide, Sigma-   gelatin, Sigma-   distilled water-   1M HCl (hydrochloric acid), BDH    Method

A 1% w/w carbomer dispersion was made in 0.1M MES buffer, adjusted toneutral and stirred until clear. Gelatin was then added to this suchthat the final concentration was between 0 and 8% w/w, depending on thedesired product and the pH kept at neutral. The system was thenconjugated using 50 mM EDAC, the reaction being quenched after stirringfor three hours at room temperature by diluting with PBS at pH 5.9 andcentrifuging washings from the partially-dehydrated carbomer conjugateuntil washings revealed no further con A in the supernatant. The totalprotein removed was calculated by assaying bulked and filtered washingsat 276 nm. The pH of the gel was then adjusted to 7.4 using 1M NaOH anda stiffened gel results than responds to temperatures above 37° C. bysoftening significantly, because of the gelatin influence.

Rheology Testing

Gels of 8% gelatin content and 2% carbomer 974 were subjected torheological testing. The appropriate tests are the oscillatory,non-destructive tests which are reported here in terms of the tan deltaor loss tangent. This parameter is the ratio of the viscous and elasticmoduli and consequently a value of unity indicates that thecontributions from each of these is equal. A value of greater than onemeans that the material has become more liquid in character. Thereforestress and frequency sweeps are done with the intention of assessing thetan delta profiles.

Results are shown in FIGS. 15 a-f. In these graphs, the profiles havebeen measured across both stress and frequency giving three-dimensionalplots that clearly indicate chymotrypsin activity in terms of raised tandeltas, particularly at higher stress values, these being very muchlower at zero enzyme content. The exception to this is at temperaturesare raised above 37° C., the temperature at which gelatin melts. Thus,at 37° C., this system would be ineffective since the material wouldalready be very softened without enzyme. Fortunately, skin temperatureis around 32° C., where the effect is as visible as at 20° C.

This test, with an enzyme very similar to stratum corneum chymotrypticenzyme (SCCE) found over-expressed in psoriatic plaque, suggests thatthe material would lose viscosity selectively over plaque and not overnormal tissue, much as it has done in these artificial conditions. Thediffusibility of drugs through the gelatin-rich phase (i.e. theinterstitial regions of the material), is related to the viscosity thereand the conclusion is that delivery of drug would be raised in areasover abnormal skin.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

While the foregoing specification has been described with regard tocertain preferred embodiments, and any details have been set forth forthe purpose of illustration, it will be apparent to those skilled in theart without departing from the spirit and scope of the invention, thatthe invention may be subject to various modifications and additionalembodiments, and that certain of the details described herein can bevaried considerably without departing from the basic principles of theinvention. Such modifications and additional embodiments are alsointended to fall within the scope of the appended claims.

1. A gel system comprising Concanavalin A and dextran gel-formingmoieties, stabilized by a network comprising a plurality of acrylicpolymer backbones, formed by copolymerizing methacrylate derivatives ofConcanavalin A and dextran, wherein 1) the polymer backbones arepermanently cross-linked through one or more dextran moieties; and 2)pendant Concanavalin A and dextran moieties are appended to thebackbones, and the pendant moieties are reversibly cross-linked, suchthat reversible binding controls viscosity of the gel system; wherein inabsence of an analyte, all permanent and reversible cross-linkages arepresent and the gel system is a viscous liquid, but in presence ofanalyte, the reversible-cross-links between the pendant moieties arereversed, effecting increasing permeability corresponding to increasingconcentration levels of analyte.
 2. The gel system of claim 1, incombination with a semi-permeable or permeable membrane.